Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-21
2004-01-06
Forman, BJ (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S243000
Reexamination Certificate
active
06673538
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the identification of groups within a species, and in particular, methods and compositions for determining a statistically significant number of different strains within a species of bacteria indicative of the species population structure as a whole in order to permit the evaluation of a vaccine target.
BACKGROUND
Bacterial infections continue to account for a considerable amount of human illness. While antibiotic therapy is clearly one of the great success stories of modem medicine, the development of antibiotic resistant strains of important human pathogens has called into question the use of antibiotics as the first line of defense against bacterial pathogens.
Vaccines to a variety of bacteria have been attempted. The best results thus far have involved vaccines directed to specific toxins of the organism (e.g. diphtheria toxoid, tetanus toxoid, etc.). Considerably less favorable results have been achieved with whole organism (“killed bacteria”) vaccines (e.g.
Bordetella pertussis, Vibrio cholerae
, etc.). Indeed, immunity induced by vaccination with killed organisms such as
V. cholerae
persists for a only a few months and therefore is of very limited value.
One important problem with current approaches to vaccine development stems from the range of variability within a species of any particular surface antigen considered as a possible vaccine target. This accounts for the fact that only a few important, new bacterial vaccines have been produced in the last 30 years (i.e. for
Haemophilus influenzae
type b, a major cause of meningitis). Moreover, development of even these recent few successful vaccines were a tedious and haphazard endeavor with little progess seen for many years.
What is needed is a more efficient approach to vaccine development. Importantly, the new approach should be one that takes into account the variability in surface antigens within a species.
SUMMARY OF THE INVENTION
The present invention relates to the identification of groups within a species, and in particular, methods and compositions for determining a statistically significant number of different strains within a species of bacteria indicative of the species population structure as a whole in order to permit the evaluation of a vaccine target. The present invention employs a method comprising the grouping of strains within a species to approximate the minimum variability in any vaccine target. This permits the evaluation of the vaccine target in a more limited number of bacterial isolates (as opposed to the two extremes of 1) using but a single isolate and 2) testing hundreds of isolates at random).
In one embodiment of the method of the present invention, the present invention contemplates analysis of the flanking sequences of one or more so-called Ribosomal RNA Operons, each comprising three genes arranged in the order 16S-23S-5S, with “spacer” DNA separating each gene (hereinafter represented by: 5′-16S-spacer-23S-spacer-5S-3′). The present invention contemplates that the analysis of these flanking sequences in a statistically significant number (e.g. greater than one hundred, and more preferably greater than three hundred, and most preferably greater than five hundred) clinical isolates of a particlar bacterial or fungal species.
It is not intended that the present invention be limited by the technique by which the flanking sequences of such operons are analyzed. In one embodiment, primers directed to these sequences can be employed in an amplification reaction (such as PCR). On the other hand, these flanking sequences can conveniently be analyzed with restriction enzymes. Specifically, the present invention contemplates digesting bacterial or fungal DNA with one or more restriction enzymes which will produce a piece of nucleic acid of which at least a portion is outside (not bounded by) the 5′ and 3′ ends of the operon. For the convenience of detecting such digestion products by gel electrophoresis, it is preferred that the digestion product (due to the relatively limited resolution level of gel electrophoresis) be at least 200 bp in size (and more preferably between 400 and 30,000 bp in size).
In one embodiment, the present invention contemplates digestion of such DNA with restriction enzymes that cut only once in the DNA encoding 16S ribosomal RNA and only once in the DNA encoding 23S ribosomal RNA. In a preferred embodiment, the present invention contemplates digestion of bacterial DNA using a single restriction enzyme which cuts only once in the DNA encoding 16S ribosomal RNA and only once in the DNA encoding 23S ribosomal RNA.
In one embodiment, the present invention contemplates a method for vaccine development, comprising: a) providing a plurality of isolates of a single bacterial species, said isolates comprising DNA; b) examining said DNA from said isolates under conditions such that a phylogenetic tree is produced defining one or more phylogenetic subsets of said isolates; and c) evaluating a vaccine target antigen in said subset of isolates for variability.
In one embodiment, the present invention contemplates a method for vaccine development, comprising: a) providing a plurality (e.g. a panel) of clinical isolates of a single bacterial species; b) isolating bacterial DNA from each of said clinical isolates under conditions such that a DNA preparations is produced for each isolate, said DNA preparation comprising DNA flanking the DNA encoding 16S and 23S rRNA; c) digesting said DNA preparations with one or more restriction enzymes under conditions such that restriction fragments are produced, said restriction fragments comprising a digestion product for each of said isolates, said digestion product comprising a portion of said DNA encoding 16S rRNA or 23S rRNA and a portion of said DNA flanking said DNA encoding 16S rRNA or 23S rRNA; d) separating of said restriction fragments (e.g. by gel electrophoresis), e) detecting said digestion products of each of said isolates; f) grouping said isolates based on the number of digestion products having identical size to define one or more subsets of isolates; g) evaluating a vaccine target antigen in said subset of isolates for variability [e.g. examining the gene(s) encoding the antigen or the gene(s) encoding essential enzymes in the biosynthesis of the antigen].
It is not intended that the present invention be limited to the method by which the results are evaluated and grouped as set forth in step (f) above. A variety of types of phylogenic analysis can be employed. What is important is to use the phylogeny of the species of interest and look for antigen-encoding conserved genes that may be important in developing a vaccine.
It is not intended that the present invention be limited by the nature of the sample. The terms “sample” and “specimen” in the present specification and claims are used in their broadest sense. On the one hand they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, cells as well as solid tissue (including both normal and diseased tissue). These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms.
It is not intended that the present invention be limited by the means of detection or the means of comparing digestion products. In one embodiment, said digestion products that are separated by gel electrophoresis are probed with a labeled oligonucleotide in a hybridization reaction.
It is not intended that the present invention be limited by the number of samples compared. A large number of clinical samples of a particular species are specifically contemplated within the scope of the present invention.
DEFINITIONS
To facilitate understanding of the invention, a number of
Forman BJ
Strzelecka Teresa
The Trustees of Boston University
Weingarten Schurgin, Gagnebin & Lebovici LLP
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